KR100536478B1 - Biodegradable collagen film used for artificial organs and method for preparing same - Google Patents

Abstract

The present invention relates to a biodegradable collagen film for producing artificial organs and a method for producing the same, specifically, preparing a collagen sponge with less than 1.0% collagen; Dipping the collagen sponge in a glucosaminoglycan (GAG) solution to form a collagen film; And it relates to a biodegradable collagen film produced by the step of crosslinking the collagen film and a method for producing the same. The biodegradable collagen film of the present invention improved the physical properties by using a low concentration of collagen solution, using a natural polymer as a substrate for the production of various artificial organs by reducing the immune response (immune response) and increase the engraftment rate It can be usefully used.

Description

Biodegradable collagen film for manufacturing artificial organs and its manufacturing method {BIODEGRADABLE COLLAGEN FILM USED FOR ARTIFICIAL ORGANS AND METHOD FOR PREPARING SAME}

The present invention relates to a biodegradable collagen film for producing an artificial organ prepared in the form of a film by crosslinking collagen and glucosaminoglycan, which is a biodegradable polymer, and a method of manufacturing the same.

Tissue engineering is the study of cell-substrate complexes by three-dimensionally culturing cells on a biocompatible substrate and using them to regenerate biological tissues and organs. The basic principle of tissue engineering is to first collect a small amount of tissue from the patient's body, separate the cells from the tissue pieces, and then proliferate the separated cells through culture in necessary amounts and incubate them in various forms of biodegradable polymer substrates for a period of time. After culturing, the cell-substrate complex is transplanted into the human body. After transplantation, cells are supplied with oxygen and nutrients by the diffusion of body fluid until new blood vessels are formed, but when blood vessels grow and supply blood in the body, the cells proliferate and differentiate to form new tissues and organs. It is disassembled and disappears.

Tissue engineering is applied to the regeneration of all organs of the human body such as artificial skin, artificial bones, artificial cartilage, artificial cornea, artificial blood vessels, artificial muscles, artificial tendons, etc. It is very important to manufacture.

The first product commercialized using tissue engineering technology is artificial skin. For example, INTEGRA ^™ (Integra LifeScience) was developed for dermal regeneration as a silicone film adhered to a collagen sponge, while Apligraf ^™ (Organogenesis) A product containing fibroblasts and keratinocytes, licensed from the US Food and Drug Administration (FDA) for the treatment of ulcers. CCS of Ortec in the United States ^? Is used as artificial skin using a sponge-like collagen matrix in which fibroblasts and keratinocytes are cultured.

In the case of wounds of large areas such as burns, the transplantation of autologous epidermis is the most desirable treatment after engraftment. However, since autologous epidermal transplantation causes lack of autologous tissue and new lesions, studies on the use of cultured autologous epithelial layers have been actively conducted since the 1980s. The treatment method using the cultured epithelial cells is currently used for the treatment of urinary and cornea in addition to the skin. However, when only the cultured epithelial cell layer is transplanted, it is easy to tear, and thus, the transplantation procedure is difficult and the engraftment rate is low. Therefore, studies to use a polyurethane sheet, a hyaluronic acid film, a collagen film, and the like to compensate for this disadvantage are actively underway.

Ponec et al. (Ponec, M. et al., Wound Repair and Regeneration 7: 214-225, 1999) reconstituted keratinocytes on porous polycarbonate or polyester membranes to reconstitute the stratum corneum and treat the enzyme with the recovery of the cultured epidermal layer. The engraftment rate was increased by shortening the time, and Wentworth et al. (Wentworth, BM et al., Burns 24: 7-17, 1998) reconstituted the epidermal layer by culturing keratinocytes on a polyurethane membrane called HydroDerm ^TM . (Valentian, Z. et al., J. Biomed Mater Res . 40: 187-194, 1998; Giampaolo, G. et al., Biomaterials 21: 2183-2191, 2000) exfoliate on a porous hyaluronic acid membrane called laserskin ^™ . The epidermal layer was reconstituted by culturing the forming cells. And, Horch et al., Tissue Engineering 6 (1): 53-67, 2000) called TissuFascie ^TM Keratinocytes were cultured in a single layer on the collagen film. In addition, Korean Patent Publication No. 1992-336 discloses a method for culturing keratinocytes in a perforated artificial synthetic membrane.

Biocompatibility is very important when culturing cells in a biodegradable polymer matrix as described above and transplanting the cell-substrate complex obtained therefrom. Therefore, studies are being actively conducted to produce a film using collagen having the best biocompatibility among the various materials.

Tiller et al. (Tiller, JC et al., Biotechnol Bioeng . 73: 246-252, 2001) described the polyethylene on collagen films prepared by acetylation or succinylation of collagen and drying 0.25% collagen solution. Glycol (polyethyleneglycol, PEG) was treated to increase flexibility and antimicrobial properties. Yamaguchi et al. (Yamauchi, K. et al., Biomaterials 22: 855-863, 2001) attached SH groups between collagen molecules, To 0.5% collagen solution was dried to prepare a film to improve the degradability for the enzyme (collagenase). Scott (R., Urological Research 12: 295-299, 1984) prepared a collagen film by adding 0.25% glycerol to 0.3% collagen solution and then drying. Korean Patent Publication No. 2003-10636 discloses a collagen film prepared by gelling 0.7-2% collagen and then drying or compressing at high pressure after preparing a sponge, and then attaching the collagen to a porous substrate.

Although many studies have described methods for preparing a film-type substrate using collagen, they all have a high concentration (2%) of collagen or polymers added to collagen molecules due to the low physical properties of collagen. have.

Therefore, the present inventors have diligently studied to develop a biodegradable polymer substrate for artificial organ manufacturing, which is less similar to biological tissues and has a tissue affinity, which is more similar to biological tissues. The present invention was completed by crosslinking only glucosaminoglycan with a low concentration of collagen solution lower than 2%) to prepare a biodegradable collagen substrate in the form of a film.

 It is an object of the present invention to provide a biodegradable collagen substrate for the preparation of artificial organs which can be transplanted into the body alone or by culturing cells and which does not cause an immune rejection reaction after transplantation.

In accordance with the above object, the present invention provides a biodegradable collagen film for producing an artificial organ prepared in the form of a film by crosslinking the collagen and glucosaminoglycan, which is a biodegradable polymer substrate, and a method of manufacturing the same.

Hereinafter, the present invention will be described in detail.

Biodegradable collagen substrate of the present invention was prepared using only biodegradable natural polymers compared to artificial synthetic substrates and natural-artificial substrates, which are conventionally used as substrates, thereby reducing the immune response that is a concern during transplantation and increasing the engraftment rate. Although the collagen solution is used, there are characteristics in improving physical properties compared to the existing collagen film.

Biodegradable collagen film for artificial organ production of the present invention

1) lyophilizing less than 1.0% collagen solution to prepare a sponge-like substrate;

2) immersing the collagen substrate in a glucosaminoglycan solution, taking it out, and drying at 4 to 20 ° C. for 24 to 48 hours to form a collagen film; And

3) may be prepared by crosslinking the collagen film.

The collagen used in step 1) has good adhesion and proliferation of tissue cells, well preserves the function of differentiated cells, and is well compatible with surrounding tissues even after transplantation in the body. Do not decompose and remain as a foreign substance.

The sponge-like collagen layer is prepared by dissolving collagen in an acidic solution at a concentration of less than 1%, preferably 0.5 to 0.7%, to make a creamy solution, and then applying it to a well plate, completely freezing in a cryogenic freezer, and lyophilizing. do. The acidic solution that can be used in the above is an aqueous solution of acetic acid or hydrochloric acid, the concentration of the acidic solution is preferably 0.01 to 0.1 M.

Collagen that may be used in the present invention includes both insoluble collagen and soluble collagen. In addition, the collagen of the present invention is preferably derived from a mammal such as a cow or from a marine organism such as the skin of a tibia fish. More preferably fish with colorless skin, most preferably flounders, for example fishes such as Seodae, Munchi flounder, Turbot, Brill can be used.

In step 2), the collagen sponge prepared as above is immersed in a glucosaminoglycan solution of 1 to 10 mg / ml, preferably 5 to 15 mg / ml, and then placed in a petri dish and 4 to 20 ° C., preferably Collagen films are prepared by drying at 4 ° C. for 24 to 48 hours, preferably 48 hours. If the drying speed is fast, the film is warped, so it is dried slowly at low temperature.

GAG, along with collagen, is one of the extracellular substrates that cells secrete, which is a major component of the substrate of the skin, and can be an indicator for measuring the activity of cells. It combines with collagen to increase strength and physical properties. GAGs that may be used in the present invention include chondroitin, chondroitin sulfate, hyaluronic acid, heparan, heparan sulfate and the like.

Step 3) is a step of crosslinking the collagen film prepared in step 2). Crosslinking can be accomplished by physical crosslinking or chemical crosslinking, which may include thermal drying under vacuum (105 ° C., 72 hours) or UV irradiation (10 W at 4 ° C., 4 hours at 254 nm intensity), or the like. And chemical crosslinking can be carried out by crosslinking with diphenylphosphoryl azide, glutaraldehyde, hexamethylene isocyanate, succinimide or carbodiimide.

Biodegradable collagen film for the preparation of artificial organs prepared according to the present invention provides an optimal environment for keratinocyte culture and secretes laminin and type IV collagen, and maintains many stem cells and enzymes for desorption from dishes after cultivation like culture epidermis. It offers the advantage of eliminating the step of processing. In addition, the biodegradable collagen film for manufacturing artificial organs of the present invention is improved in the physical properties by producing a film with a low concentration of collagen solution, and because only biodegradable natural materials are used, it is excellent in biocompatibility, thereby reducing the inflammatory response during transplantation. Therefore, the collagen film for artificial organ production of the present invention can be usefully used as a substrate for manufacturing various artificial organs such as artificial skin, artificial cornea, artificial mucosa, and the like. In addition, the collagen film can also be used as a cell culture substrate of the test kit for the screening of in vitro toxicity and bioactive substances.

As an example of such an application range, the artificial epidermal layer was formed by culturing keratinocytes on a biodegradable collagen film according to the preparation method of the present invention, and then transplanted into nude mice, and the therapeutic effect was higher than that of the epidermal layer cultured only with cells. It confirmed that it is better.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Collagen Film

Type I collagen was dissolved in 0.05 M acetic acid to prepare a final concentration of 5 mg / ml solution, and then 1,500 μl was applied to a 12-well plate, frozen at -85 ° C. in a cryogenic freezer for at least 12 hours, and then vacuum lyophilizer (80 ° C.). Collagen sponge was prepared by freeze drying for 36 hours in SFDSM 06, 3). The collagen sponge in the lyophilized porous form was immersed in a glucosaminoglycan solution at a concentration of 5 to 15 mg / ml, taken out, placed in a dish, and dried at 4 ° C. for 48 hours to prepare a collagen film. The collagen film thus prepared was soaked in 0.25% glutaraldehyde solution for 24 hours for crosslinking and washed with tertiary distilled water for 7 days (0.5% Coll; experimental group).

In the comparative group, a mixed solution of GAG pre-added to 0.5% collagen solution was applied on a dish about 5 ml, and dried at room temperature for 3 days to prepare a collagen film (0.5% Coll-GAG), and a 2% collagen solution was used as a control. Collagen film (KOKEN, Japan) prepared according to the general drying method was used.

1A shows a collagen film (0.5% Coll; A) prepared according to the present invention, a collagen film (0.5% Coll-GAG; B) made with a collagen solution containing GAG, and a collagen film made with a 2% collagen solution ( 2% Coll; C) As a result of H / E staining of the films to confirm their structural differences, the collagen film of the present invention ( FIG. 1B ) showed a continuous and regular structure, whereas the collagen film of the comparative group ( FIG. 1C ) had pores . It can be seen that it has a discontinuous structure, which forms an opaque portion in B of FIG. 1A . When the film was prepared by a general drying method using a 0.5% collagen solution, the film was not properly formed.

Example 2 Cultivation of keratinocytes on collagen film

Primary cultures of keratinocytes were performed according to enzymatic degradation (EK Yang, Artificial Organs 24: 7-17, 2000). First, to separate epidermis and dermis, 3 mg / ml (1,000 U) of collagenase in which foreskin was dissolved in calcium acetate buffer (130 mM NaCl, 10 mM Ca acetate, 20 mM HEPES, pH 7.2) 1 mg / ml (1.5 U) dispase solution dissolved in PBS and an enzyme solution was immersed in a 1: 1 mixed enzyme solution and treated at 4 ° C. for 16 hours to separate epidermis and dermis. It was. The epidermis was separated into 0.05% trypsin enzyme solution and treated at 37 ° C. for 15 minutes, followed by DMEM (Dulbecco's modified Eagle's medium, GIBCO 12100-038) medium with 10% fetal bovine serum (FBS). After diluting with centrifugation, keratinocytes were obtained. The keratinocytes thus obtained were incubated for 10 days in a medium containing DMEM and F12 3: 1 containing serum-free medium or 10% serum in an incubator at 37 ° C. and 5% CO ₂ .

The collagen film (0.5% Coll) and the control film (0.5% Coll-GAG and 2% Coll) prepared in Example 1 were placed on the bottom of the dish, and keratinocytes were prepared at a concentration of 5 × 10 ⁵ cells / cm 2. After inoculation on the film was placed in the incubator for 3 hours. Subserge culture was performed for about 5 days, and then air-liquid interface culture was performed on the stainless steel net. At this time, epidermal growth factor (EGF) in the medium was removed and cultured for about 1 week to obtain a layered epidermal layer.

Figure 2 is a result of incubation for 4 days by inoculating keratinocytes to confirm whether the collagen film of the present invention has a cell compatibility. As shown in FIG. 2 , the cells inoculated on the collagen film grew well to confirm that the collagen film of the present invention had excellent cell compatibility. The white, cloudy area around it indicates that the cells are stratified.

Example 3 Dyeing

In order to check whether the collagen film of the present invention prepared as described above has cell compatibility, the cells were cultured as in Example 2, and then H / E (hematoxylin eosin) staining and immunochemical staining (laminin, type IV collagen, cytosine). Keratin-10 and Ki-67 staining). Histological examination was conducted with reference to a biopsy studies (clinical pathology professor at the National Assembly, considered Medicine 1992) and histologic staining special book (gimjuho, Kwang Publisher 1993).

(1-1) H / E dyeing

The cell cultured films were fixed with 4% formaldehyde solution, dehydrated with alcohol, cleared with xylene, and then cut from the embedding in paraffin. Hematoxylin / eosin , H / E) and observed with an optical microscope.

As a result, as shown in Figure 3 , it can be seen that the cell is most stratified on the collagen film (A) prepared according to the present invention, the polarity of the basal layer cells is also well maintained. This indicates that the mass transfer of a substance such as a medium or a nutrient in the collagen film prepared according to the present invention is better than that made with high concentration collagen (C). In addition, if the surface structure of the film is discontinuous and irregular (B) it can be seen that the formation of the basal cell layer is not properly made.

(1-2) laminin staining

Formalin-fixed paraffin embedded tissue was made into 4 μm thick tissue sections, and then attached to a slide coated with poly-L-Lysin (Sigma, St Louis) and dried. Tissue sections were deparaffinized with xylene and then treated with 100, 95, 90, 85 and 70% alcohol for 5 minutes each to hydrate with distilled water (rehydration) and wash. In order to inhibit the endogenous peroxidase activity was treated with 3% H ₂ O ₂ for 10 minutes and washed with 0.05 M TBS (triphosphate buffered saline) buffer. Proteinase K was treated for 30 minutes at room temperature to increase tissue antigen expression, and pre-blocked for 5 minutes with 5% skim milk (non-fat milk) to suppress nonspecific reactions. blocking) and washed thoroughly with distilled water. As a primary antibody, a monoclonal antibody (DAKO) obtained from a mouse was used, which was diluted 1:50, reacted with the sample for 1 hour, and washed 3 to 4 times with 0.05 M TBS for 5 minutes. . Thereafter, biotin-attached secondary antibodies (DAKO, Denmark, LSAB2 kit) were treated for 20 minutes, washed with 0.05 M TBS, and treated with streptavidin-HRP hydrogen peroxide for 20 minutes. The resultant was washed with 0.05 M TBS, and then developed for 10 minutes using diaminobenzidine (DAB) as a labeling reagent. After the color reaction, counterstaining was performed for 3 minutes with Harris hematoxylin solution. After the sample was dehydrated and sealed, it was observed with an optical microscope. Through the staining can be confirmed newly formed laminin.

As a result, as shown in FIG . 4 , laminin (brown) was secreted only in the collagen film (0.5% Coll, A) prepared according to the present invention, and in 0.5% Coll-GAG (B) and 2% Coll (C) Laminin was found not to be secreted. These results indicate that the collagen film of the present invention provides the most suitable culture environment for the cells. Laminin and type IV collagen are the main components of the basement membrane, and the base layer is well formed when the keratinocytes are cultured in advance. However, the collagen film according to the present invention secretes the laminin by adhering the cells without coating the extracellular matrix. It was confirmed.

(1-3) Type IV Collagen Staining

Immunohistochemical staining for type IV collagen was performed in the same manner as for laminin staining, except that anti-type 4 monoclonal antibodies (NeoMarkers) obtained from mice were used as primary antibodies. Through this staining, newly formed type IV collagen can be observed.

As a result, as shown in FIG . 5 , type IV collagen (brown) was continuously secreted from the collagen film (A) prepared according to the present invention, and only a small amount was secreted from the control collagen film (C). However, type IV collagen could not be observed in 0.5% Coll-GAG (B). It can be seen from this that the collagen film according to the present invention provides the most suitable culture environment for the cells.

(1-4) Cytokeratin-10 (CK-10) Staining

Immunohistochemical staining for cytokeratin-10 was carried out in the same manner as for laminin staining except that a monoclonal antibody (anti-CK-10 monoclonal antibody, Biogenex) obtained in mice was diluted 1:40 as the primary antibody. It was. The differentiation of cells can be observed through the staining.

As a result, as shown in FIG . 6 , cytokeratin-10 (brown) was expressed in the collagen film (A) and the comparative group collagen film (B) prepared according to the present invention, but the cytokeratin-in the control collagen film (C). 10 was not expressed. This is because the mass transfer of the collagen film according to the present invention is made better than that made of high concentration collagen, and the continuous and flat surface stabilizes the formation of the basal cell layer to stably support the differentiation of the superstructure (stratum corneum). do.

(1-5) Ki-67 Dyeing

Ki-67 immunochemical staining on epithelial stem cells was treated with ACE (3-amino-9-ethylcarbazole) instead of protease K and anti-Ki-67 polyclonal rabbit anti- human, DAKO), but the same method as laminin staining. Epithelial stem cells in the differentiation or proliferation can be observed through the staining.

As a result, as shown in Figure 7 , when the cells are cultured on other substrates, the stem cells are not stained due to aging, but the cells cultured on the collagen film prepared by the present invention, the stem cells (arrows) are well left to the cells It can be seen that it provides good culture conditions.

Example 4 Animal Transplantation Test

The artificial epidermis cultured keratinocytes for 2 weeks on the collagen film prepared according to the present invention and culture epidermis (CEA) cultured in dish only keratinocytes as a control in a nude mouse. First, the back of the nude mouse was cut into the entire layer with a diameter of 1.5 cm, followed by implantation of culture epidermis (CEA) and artificial epidermis. Four days, two weeks, four weeks, and eight weeks after transplantation, the biopsy was performed, and histological staining was performed to confirm the movement of cells from the surrounding tissues and the regeneration of the dermis and epidermis.

8A is a photograph of a transplant site such as one week after implanting artificial skin in nude mouse, and it can be seen that the artificial epidermis (left) is less contracted than the culture epidermis (right). After implantation of the artificial skin, the smaller the contraction of the affected area, the less scar or fibrosis. Cultured epidermis is currently used in a lot of patients for epidermal regeneration, but it can be expected that the artificial epidermis according to the present invention exhibits a better therapeutic effect by inhibiting contraction. B and C of FIG. 8 are biopsy photographs of the second week of the artificial epidermis and the culture epidermis, respectively, but the artificial epidermis (B) is composed of the overall dermal regeneration, but the culture epidermis (C) still has a gap in the dermis. You can observe the slow progression. From the above results, it was confirmed that the collagen film prepared according to the present invention provides a good environment for the proliferation and differentiation of cells and is effective for regeneration of affected areas after transplantation.

Example 5 Analysis of Physical Properties of Collagen Film

<5-1> Tensile strength test

The collagen film (0.5% Coll) and the control film (0.5% Coll-GAG and 2% Coll) prepared according to the present invention were cut to a size of 5 × 10 mm, respectively, and then tensile strength measuring instrument (Universal testing machine, HOUSFIELD, UK) The breaking load was measured by pulling at a rate of 1 mm / min.

As a result, as shown in Figure 9 , the collagen film (0.5% Coll) prepared according to the present invention showed a stronger tensile strength than the control collagen film (0.5% Coll-GAG and 2% Coll). It was confirmed that this is because the position of the glucosaminoglycan bound to the structure of the collagen molecule is changed according to the manufacturing process sequence, thereby changing the stability of the structure. In addition, the collagen film of the present invention showed a somewhat higher tensile strength than the collagen of 2% concentration is the result of glucosaminoglycans are more stably crosslinked than when the collagen is produced only to improve physical properties.

<5-2> Swelling ratio test

The collagen film (0.5% Coll) and the control film (0.5% Coll-GAG and 2% Coll) prepared according to the present invention were cut into sizes of 5 × 10 mm, respectively, and placed in phosphate buffer (PBS) at room temperature for 48 hours. After treatment for a while to remove the moisture on the surface immediately weighed. The water content was calculated by dividing the mass of the film soaked in buffer with the mass of the dried film.

As a result, as shown in Figure 10 , the difference in moisture content according to the manufacturing process and the concentration of the collagen solution between the collagen film (0.5% Coll) and the control film (0.5% Coll-GAG and 2% Coll) prepared according to the present invention Was found to be absent. When glucosaminoglycan binds to the collagen molecule, it tends to be hydrophobic.However, the water content is similarly measured, indicating that the low concentration (0.5%) of the collagen has a similar hydrophilicity to the high concentration (2%) of the collagen. have.

As described above, the biodegradable collagen film for manufacturing an artificial organ according to the present invention has excellent physical properties and biocompatibility, but has a low risk of an immune response that is a concern during transplantation, and thus, various artificial artificial skin, artificial cornea, artificial ureter, etc. It can be usefully used as a substrate for organ preparation.

FIG. 1A is a collagen film prepared by preparing a collagen matrix according to the present invention and then crosslinking glucosaminoglycan (GAG) (A; 0.5% Coll) with a collagen film (B; 0.5% Coll-GAG), and collagen solution of 2.0% concentration of the collagen film (C; 2% Coll) prepared according to the general drying method, the picture taken

Figure 1b is the result of H / E staining of the collagen film (0.5% Coll) prepared according to the present invention,

1C is a result of H / E staining of a collagen film (0.5% Coll-GAG) prepared using a sponge containing GAG as a control,

2 is a photograph (× 200) observed under a microscope by culturing keratinocytes on a collagen film prepared according to the present invention.

3 is a result of performing H / E staining to confirm the stratification after culturing keratinocytes on various collagen films,

A: 0.5% Coll, B: 0.5% Coll-GAG, C: 2% Coll

4 is a result of laminin immunochemical staining to confirm the laminin secretion after culturing keratinocytes on various collagen films,

A: 0.5% Coll, B: 0.5% Coll-GAG, C: 2% Coll

5 is a result of performing type IV immunochemical staining to confirm the type IV collagen secretion after culturing keratinocytes on various collagen films.

A: 0.5% Coll, B: 0.5% Coll-GAG, C: 2% Coll

6 is a result of cytokeratin 10 (CK-10) immunochemical staining in order to confirm the differentiation of cells after culturing keratinocytes on various collagen films,

A: 0.5% Coll, B: 0.5% Coll-GAG, C: 2% Coll

7 is a result of performing Ki-67 immunochemical staining to check the presence of stem cells after culturing keratinocytes on the collagen film (0.5% Coll) prepared according to the present invention,

8 shows the results of culturing keratinocytes on various collagen films and transplanting them into animals.

A: Photograph showing contraction of affected part 1 week after transplantation

B: Tissue examination photograph 2 weeks after artificial epidermal transplantation in which keratinocytes were cultured on the collagen film (0.5% Coll) of the present invention

C: Tissue examination photograph 2 weeks after transplantation of cultured epidermis containing only keratinocytes

9 is a graph measuring the tensile strength of the collagen film prepared according to the present invention,

10 is a graph measuring the swelling ratio of the collagen film prepared according to the present invention.

Claims (11)

1) freeze drying less than 1% of the soluble collagen solution to prepare a substrate in the form of a sponge; 2) immersing the collagen substrate in a glucosaminoglycan solution, taking it out, and drying at 4 to 20 ° C. for 24 to 48 hours to form a collagen film; And 3) cross-linking the collagen film. A biodegradable collagen film for artificial organ production. delete The method of claim 1, Collagen film, characterized in that the collagen is derived from mammals or marine life. The method of claim 3, wherein Collagen film, characterized in that the collagen is derived from cattle or tibia fish. The method of claim 1, Glucosaminoglycans are selected from the group consisting of chondroitin, chondroitin sulfate, hyaluronic acid, heparan and heparan sulfate. 1) freeze drying less than 1% of the soluble collagen solution to prepare a substrate in the form of a sponge; 2) immersing the collagen substrate in a glucosaminoglycan solution, taking it out, and drying at 4 to 20 ° C. for 24 to 48 hours to form a collagen film; And 3) comprising the step of crosslinking the collagen film, a method for producing a collagen film. The method of claim 6, The method of claim 1, characterized in that the concentration of the collagen solution is 0.5 to 0.7%. The method of claim 6, Method for producing a glucosaminoglycan solution in step 2) is 5 to 15 mg / ㎖. The method of claim 6, The process according to claim 3, wherein the crosslinking is by physical crosslinking or chemical crosslinking. The method of claim 9, Physical crosslinking is performed by thermal drying under vacuum or UV irradiation. The method of claim 9, Wherein the chemical crosslinking is crosslinking with a compound selected from the group consisting of diphenylphosphoryl azide, glutaraldehyde, hexamethylene isocyanate, succinimide and carbodiimide.